Despite recent therapeutic advances in advanced melanoma, Stage IV melanoma remains an incurable disease with poor survival. New targets for melanoma therapy must be fully explored to lengthen survival in Stage IV patients and possibly improve cure rates in early stage patients. Autophagy, the process by which cells dispose of damaged organelles and recycle nutrients to fuel further growth, has been identified as a tumor survival mechanism. It appears to play a particularly important role in melanoma cell survival and is a promising new target in melanoma therapy. We have developed a number of clinical trials assessing the safety and preliminary activity of the first generation autophagy inhibitor hydroxychloroquine (HCQ) in combination with other anticancer drugs that induce autophagy in many malignancies. The most promising results so far were in a phase I trial of the mTOR inhibitor temsirolimus and HCQ in patients with advanced melanoma. While preliminary results show some promising antitumor activity, incorporating effective autophagy inhibitors into treatment of melanoma patients is limited by 3 factors: a) tools to monitor autophagy modulation in patients are limited b) the mechanisms of resistance to autophagy inhibition have not been identified, and c) novel, more potent second generation autophagy modulators need to be developed and tested. Using powerful proteomics techniques paired with novel autophagy modulators, patient derived xenograft models and clinical trials, we will test the hypothesis that autophagy inhibition can significantly improve the antitumor activity of PI3K/mTOR inhibitors in melanoma. To test this hypothesis and address the shortcomings listed above, thereby moving the field forward, we propose the following specific aims: Aim 1. Identify tumor-secreted protein pharmacodynamic markers (PD) of autophagy modulation and identify signaling pathways associated with acquired resistance to first generation mTOR and autophagy inhibitors. Aim 2. Determine autophagy modulation and anti-melanoma activity of the combination of a PI3K/mTOR inhibitor and a second generation autophagy inhibitor. Upon completion of this project we expect to establish novel pharmacodynamic markers and markers of resistance to autophagy modulation. We also expect to determine the comparative efficacy of second generation versus first generation autophagy inhibitors. Our studies will provide the tools and knowledge to develop targeted therapy combinations involving mTOR and autophagy inhibitors that could significantly improve the survival of melanoma patients. The knowledge gained would expedite the development of autophagy modulators, which represent a new class of drugs that have the potential to improve survival and cure rates in melanoma patients.